The research goal is to ensure the safety, purity and potency of vaccines and other biological products through research relating to the development of new or improved accurate, validated, qualitative and/or quantitative methods for the determination and/or characterization of the chemical preservatives, stabilizers, inactivators, adjuvants, residual moisture, protein and other chemical constituents of vaccines and biological products regulated by CBER and subject to license or release action. Improved methodologies include chromatographic, thermogravimetric, spectroscopic and mass spectomeric methods of analysis. Determination of residual moisture in freeze-dried biological products. The goal of this project is to develop new or improved methods for the determination of moisture in biological products. A low residual moisture is necessary for the stability, viability and potency of the freeze-dried biological product. The residual moisture of freeze-dried biological products was first determined by the gravimetric or loss-on-drying method utilizing phosphorus pentoxide and vacuum at room temperature. This method has been optimized. For samples with uncomplicated thermogravimetric analysis (TG) curves, TG results have been shown to correlate with coulometric Karl Fischer results. Karl Fischer and TG moisture results may be different from the gravimetric moisture result for the same freeze-dried product due to the fact that different types of moisture (physically adsorbed or chemically bound moisture) are being measured. The thermogravimetric method has been used to determine the moisture content of Group A and Group C Meningococcal Polysaccharide bulks at levels of 5% to 25% moisture. Thermogravimetric/mass spectrometry (TG/MS) identified the TG transition corresponding to the loss of residual moisture in vaccines that have complex TG curves. Thermogravimetry provides precise heating conditions and weight loss information at specified temperatures while mass spectrometry identifies volatile compounds evolved during the weight loss process. A new TG/MS interface applicable to this analysis has been developed in our laboratory. The glass tubing interface connects the quartz combustion tube of the TG to the jet separator of the mass spectrometer. This interface allows continuous monitoring of the ion intensities of mass peaks m/e=18 (water) and m/e=44 (carbon dioxide) for the determination of residual moisture in freeze-dried biological products. Data has been collected clarifying thermograms for both Giant Short Ragweed Allergenic Extracts as well as Limulus Amebocyte Lysate Haemophilus b. Polysaccharide Conjugate Vaccines and other products such as Allergen Patch Test. A new TG/MS capillary interface has been developed and applied to moisture analysis for AHF and BCG vaccine. A method is being researched that will determine moisture in space above freeze-dried cake in the vial. This vapor pressure moisture methodology is being applied to the study of the redistribution of moisture between cake, head-space and stopper or head-space and cake over time. A new aspect of this project involves correlating residual moisture values calculated thermodynamically with values obtained experimentally. Near infrared Spectrometry (NIR) is being evaluated for its application to moisture determination in the freeze-dried vaccine final container. Analysis and characterization of mercury in injectable products. The goals of this project include: (1) the development of methodology for the quantitative analysis of mercury in various biological products (for example, influenza virus vaccine and immune serum globulin) resulting from the use of mercurial preservatives, (2) develop methodology for the quantitative analysis of the thimerosal molecule and any of its degradation products, (3) determine stability of mercury and thimerosal in various products, and (4) determine different mercury species present in various products containing thimerosal such as Immune Serum Globulin, DTP Vaccine, etc. Cold vapor atomic absorption spectrophotometric methodology has been developed to determine the total mercury resulting from mercurial preservatives such as thimerosal, phenylmercuric nitrate and phenylmercuric borate in various injectable biological products. Validation studies for total mercury have been completed for each of the major product types. The cold vapor atomic absorption spectrophotometric procedure used with this sample digestion procedure yields highly accurate and precise results for total mercury in these samples matrices. Thimerosal stability studies are being conducted with a number of different biological products. Thimerosal has been separated from one of its degradation products, thiosalicyclic acid, by two reverse phase liquid chromatographic procedures. Liquid chromatography with an amperometric detector polarography or gas chromatography/mass spectrometry will be explored as methods for the quantitative and/or structural characterization of the thimerosal and its degradation products. Liquid chromatography combined with inductively coupled argon plasma emission spectrometry/mass spectrometry (LC-ICP/MS) has been used to quantitate thimerosal in diluent, a Tetanus Toxoid Adsorbed and an Influenza Virus Vaccine. Work is being done on immune serum globulin by this technique. Methods are being developed to detect mercury in vaccines in which mercury has been minimized to meet current safety standards for the use of thimerosal in childhood vaccines. Instrumentation is being developed to lower the detection limit of the method. Determination of trace metals in injectable biological products. The objectives of this project are to develop and validate methodologies for the determination of trace metals in injectable biological products. This includes trace metals present as residuals of the manufacturing procedure, those present as impurities and those that are chemical constituents of biological products whose concentration is vital to product stability, efficacy or safety. Methodology used includes atomic absorption spectrometry, inductively coupled argon plasma emission spectrometry, inductively coupled argon plasma mass spectometry and ion chromatography. An interagency agreement with NIST resulted in data which gives the trace metal profile for a number of biological products. This survey identified high levels of aluminum in several albumins from one manufacturer. Toxicologists at FDA evaluated the data collected. A new objective involves evaluating the trace metal leachates from several types of glass vials including vials lined with silican dioxide coatings and glass vials fabricated with cerium. An ICP method has been developed for phosphorus in Haemphilus b Conjugate Vaccine. Work has been done on the determination of low levels of copper in Mega 1 and Mega 2 international standards for Antihemophilic Factor. Experiments in Radiation Sterilization. The goal is to determine whether radiation sterilization is able to be used with biological products. It has been approved for use on certain containers and diluents in the past. Studies are being conducted on a cross-section of biological product types using gamma irradiation (cobalt-60), electron beam and x-ray. Emphasis has been placed on childhood vaccines since alternatives to thimerosal need to be explored. Analysis and characterization of organic chemical constituents of injectable products. The project is led by Anthony Ciavarella. This research focuses on the quantitative analysis and characterization of the chemical components in vaccines including 2-phenoxyethanol. 2-Phenoxyethanol is used as a preservative in vaccines which include Hepatitis, Polio, and DTaP(Diphtheria and Tetanus Toxoids and Acellular Pertussis). To ensure the proper amount of this preservative, we have modified the USP<341> Antimicrobial Agents method to use in the quantitation of 2-phenoxyethanol. Current work includes the validation of this packed column GC method under ICH guidelines which is nearing completion. Future research will involve the development of a capillary GC method resulting in greater accuracy and column efficiency. Analytical methods for the determination of transmissible spongiform encelphalopathy (TSE) agent infectivity in differentiated neuronal cell cultures. Led by Alfred Del Grosso. This work had involved the determination of acetylcholinesterase and tyrosine hydroxylase activities and related methods development in collaborative support of project number Z01 BR05008-02 LMD, "Potential assay of transmissible spongiform encephalopathy (TSE) agent infectivity in differentiated neuronal cell cultures". Acetylcholinesterase determinations were performed by the method of G. Ellman (Biochem. Pharm. 7, 88-95, 1961). Tyrosine hydroxylase activity is measured by determination of the enzyme product DOPA (dihydroxyphenylalanine) by HPLC. In this procedure, incubation was performed in the presence of saturating concentrations of tyrosine substrate and 6-methyltetrahydropteridine cofactor; DOPA product was determined by reverse phase HPLC with amperometric electrochemical detection after bulk extraction with acidic alumina. In FY00 and 01, the method was modified based on the studies of D. Hooper in which signal-to-noise levels were improved with the addition of glycerol to reduce blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. Determination of total protein, for the purpose of normalizing enzyme activities to the concentration of cell culture, is being performed by a micro BCA (bichoninic acid) procedure. Determinations of acetylcholinesterase activity in scrapie inoculated PC12 cell culture have not to date shown a significant difference in activity from those obtained from un-inoculated cultures. These results are to be described along with modifications to the chromatographic determination of tyrosine hydroxylase activity. Further activity has been suspended with the retirement of Jeanette Ridge, PI for project Z01 BR05008-02. Chromatographic determination of chemical components of biological products. Led by Alfred Del Grosso. The objectives of this project are to develop and validate methodologies for chemical components of biological products whose concentration is vital to product stability, efficacy or safety. These include: 1) phenol used as an antimicrobial preservative in multi-use parenterals such as allergenic extracts and bacterial vaccines, 2) glycerin used in allergenic extracts as a preservative and/or stabilizer, 3) 2-phenoxyethanol used as a preservative in inactivated poliovirus vaccine and combined bacterial vaccines, 4) formaldehyde used as an inactivating agent in influenza virus vaccine, hepatitis B vaccine and other products, 5) chloride in human serum albumin and dextran volume expanders, 6) histamine in positive skin test control, 7) organic, natural product and complex synthetic mixture components of allergen patch tests. Current work in progress includes the following methods development and validation activities: 1) The complete validation of CBER gas chromatographic methods based on USP <1225> "Antimicrobial Agents - Content" for 2-phenoxyethanol. Under A. Del Grosso's direction, A. Ciavarella has validated this procedure for accuracy, precision and specificity. An evaluation of robustness and ruggedness parameters is on-going. Future plans will include the evaluation of wide-bore capillary columns to provide faster analysis and enhanced specificity through higher chromatographic efficiency. 2) An HPLC procedure for glycerin has provided lower analytical results relative to potentiometric titration when applied to food allergenic extracts. Analytical recoveries by the different techniques are being compared along with procedural modifications to enhance recovery. 3) The development of a procedure for monosaccharide composition analysis of neutral sugars in purified proteins. Preliminary results using a procedure involving selected ion monitoring capillary gas chromatography of per-trimethylsilylated saccharides indicate that composition analysis from 10 micrograms of protein, representing ca. 200 nanograms total carbohydrate should be feasible. 4) The evaluation of gas chromatography with pulsed discharge detection as a potential procedure for low levels of formaldehyde in vaccine products. Identification and quantitation of impurities and residual manufacturing agents in biological products. Led by Alfred Del Grosso. The objectives of this project are to develop and validate methodologies for chemical impurities and residual manufacturing agents in biological products whose presence may affect product safety or efficacy. These include: 1) residual glutaraldehyde used as inactivating or conjugating agent in vaccines, 2) residual tetrachloroethylene in pollen allergenic extracts and allergenic source materials, 3) inactive components of crude allergenic extracts and allergen patch test materials and 4) lipopolysaccharides (endotoxins) in allergenic extracts and vaccines. Current work in progress, or that performed in the past year includes: 1) The determination of perchloroethylene content in allergenic extracts and pollen source materials. Perchloroethylene is used in the processing of pollen source materials for allergenic extracts. A dynamic headspace gas chromatographic method for tetrachloroethylene in allergenic extracts has been developed and validated, data has been obtained for a representative sampling of final products. The determination of this same compound in allergenic source materials by gas chromatography/mass spectrometry has been validated and data on a representative sampling of pollen source materials has been collected. 2) An HPLC method for the determination of glutaraldehyde, involving pre-column derivatization with p-nitrobenzyl-hydroxylamine and UV detection has been developed and validated as a limits test at 100ppb. 3) A recently acquired ion-trap HPLC/mass spectrometer will be used along with gas chromatography/mass spectrometry to develop a general procedure for impurities screening in a variety of products as well as the characterization of low-molecular weight compounds in allergenic extracts and allergen patch test products. 4) Several chromatographic and mass spectrometric techniques are being evaluated for the determination of lipopolysaccharide content of allergenic extracts and vaccines based on characteristic 3-hydroxy fatty acid marker compounds. Determination of nitrogen content (protein content) of vaccines and other biological products. Led by Nora Etz. This study was initiated with the following objectives: (1) to standardize the protein nitrogen unit PNU method for the determination of the concentration of allergenic extracts, (2) to determine the stability of the allergenic extract PNU value throughout the dating period, (3) to determine between laboratory reproducibility for assayed PNU values, and (4) to improve the detection limit for the determination of nitrogen and decrease analysis time. Parameters were optimized for the PNU precipitation procedure for aqueous, freeze-dried, glycerinated and alum precipitated allergenic extracts. The stability study indicated stability for PNU values when the products tested have been stored at a constant 2-8oC. Although the allergens lose their reactivity with time, the PNU value does not change significantly. It is an estimate of the concentration of a freshly prepared allergenic extract. The collaborative study of the optimized PNU precipitation procedure consisted of the analysis of six samples in duplicate by six laboratories using the CBER Kjeldahl methodology. Methodology is being studied to determine the protein content in Anthrax Vaccine, Typhoid Vaccine, Cholera Vaccine, Influenza Virus Vaccine and other vaccines such as Hepatitis B Vaccine and Hepatitis C100-3 Antigen in which protein measurement by the Lowry method would be subjected to interferences by compounds which are present such as SDS, Tris, EDTA, and thiol reagents (DTT and thimerosal). A study of the above methods is underway. This project incorporates FY2002 projects 1Z01BR004002-27, 1Z01BR004003-25, 1Z01BR004004-24, 1Z01BR004005-17, 1Z01BR004006-17, 1Z01BR004007-15, 1Z01BR004008-15, and 1Z01BR004010-03.